Rothmund Thomson Syndromes (RTS) is a rare disease associated with genome instability, predisposition to cancer, skin and skeletal abnormalities, and some features of premature aging. The disease is caused by mutation in RECQL4, a putative helicase of the RecQ family. To understand the mechanism of this disease, we have purified a human RECQL4 complex and identified its associated components. The bulk of RECQL4 was present in cytoplasmic extracts of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200 kD proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins that promote protein turnovers. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis, and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. Our data are consistent with RECQL4 and ubiquitin ligases of the N-end rule pathway contributing to maintaining genomic stability. We have also purified complexes containing RECQL5, another RecQ helicase involved in maintaining genome stability. Deficiency of RecQL5 in C.elegans reduces life-span by 37%, and inactivation of this gene in mice results genomic instability and increased cancer risks. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known as transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 must have both helicase activity and association with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. Our data suggest that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination. We found that RecQL5 also interacts with several other molecules important for DNA replication and repair. These include the RAD51 recombinase and PNCA (a clamp for DNA polymerase). We have mapped their interaction domains within RecQL5, and shown that mutants that are inactivated of these interactions have reduced capacity in suppressing SCE and resist campothecin-mediated cell killing. These studies suggest that RecQL5 participates in protecting genome integrity through multiple mechanisms. We specifically characterized the RAD51-interaction domain of RecQL5 in collaboration with Patrick Sung's group. We found that this domain resembles the BRC repeat, which is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein) that promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. We named this new domain in RecQL5 as BRCv. We demonstrate that BRCv possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5. In a study of the origin of DNA damage, we participated in a collaborative project with Dr. H. Chengs group to study how mitochondria produce reactive oxygen that can modify DNA. We collaborated with other colleagues to investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQL4 is recruited to origins at late G(1), after ORC and MCM complex assembly, while RECQ1 and additional RECQL4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and to a greater extent after RECQL4 depletion. Depletion of RECQ1, though not that of RECQL4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQL4 associate with the human DNA replication complex and have distinctive roles in DNA replication initiation and replication fork progression in vivo. Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder with prominent skeletal, renal, immunological, and ectodermal abnormalities. It is caused by mutations of SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response protein. Recent studies suggest that SMARCAL1 has DNA anealing acitivity, interacts with Replication protein A (RPA), promotes replication fork restart, and participates in DNA damage response. These features resemble those of RecQ helicases. To determine the relationship of the helicase function of SMARCAL1 to the SIOD phenotype, we collaborated with Dr. Boerkoel's group and profiled the cancer prevalence in SIOD and assessed if defects of nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), respectively, explained the ectodermal and immunological features of SIOD. Among 71 SIOD patients, three had non-Hodgkin lymphoma (NHL) and one had osteosarcoma. We did not find evidence of defective NER or NHEJ; however, Smarcal1-deficient mice were hypersensitive to several genotoxic agents. Also, CPT-11, etoposide, and HU caused decreased growth and loss of growth plate chondrocytes. These data, which identify an increased prevalence of NHL in SIOD and confirm hypersensitivity to DNA damaging agents in vivo, provide guidance for the management of SIOD patients.